Method and apparatus for calibration of instruments that monitor the concentration of a sterilant in a system

ABSTRACT

A method and apparatus for calibrating a sensor for determination of the concentration of a sterilant, e.g., hydrogen peroxide vapor, in a sterilization system. This invention provides a method for calibrating a sensor that is used for measuring the quantity of a sterilant in a system for delivering the sterilant, the method comprising the steps of: 
     (a) generating reference calibration data, the reference calibration data showing a mathematical relationship between a measurable parameter, e.g. voltage, and a quantity of the sterilant, e.g., parts of sterilant per million parts of air (ppm), for a plurality of sensors; 
     (b) generating sensor calibration data, the sensor calibration data showing a mathematical relationship between the measurable parameter and the quantity of the sterilant for an individual sensor; and 
     (c) normalizing the sensor calibration data to compensate for the difference between the measurable parameter for the reference calibration data and the measurable parameter for the sensor calibration data, whereby data obtained by the individual sensor can be used to accurately determine the quantity of sterilant in the system.

This application is a division of U.S. Ser. No. 10/196,036, filed Jul.16, 2002, now U.S. Pat. No. 6,581,435, which is a division of U.S. Ser.No. 09/784,710, filed Feb. 15, 2001 now U.S. Pat. No. 6,612,149.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for calibration of instruments. Moreparticularly, this invention relates to a method for calibration ofinstruments that monitor the concentration of a sterilant, e.g.,hydrogen peroxide, in a system.

2. Discussion of the Art

Aseptic processing of consumable products, such as nutritional compoundsand food products, is typically effected by separate sterilization ofthe products and the containers within which the products are packaged.Subsequent to sterilization, the sterilized products are placed insterilized containers and sealed in a sterile environment for shipment,storage, and use.

Sterilization of such containers, which may include sterilization ofseparate closures as well, can be performed efficiently by use of asterilant, such as hydrogen peroxide (H₂O₂) vapor, prior to theintroduction of the desired sterilized products into the containers. Insuch a process, the containers are introduced into a sterilizationapparatus in which the containers are flushed with hydrogen peroxidevapor. The containers are subsequently flushed with warm air or anyother fluid suitable for achieving desirably low levels of residualhydrogen peroxide. This general procedure is highly effective inachieving sterilization of the containers and can be performed on anyother articles that will come into contact with the material to beintroduced into the containers.

Notwithstanding the effectiveness of sterilization by hydrogen peroxide,accurate monitoring of concentration levels of hydrogen peroxide vaporcan be problematic. Problems in monitoring the concentration of hydrogenperoxide vapor result in part from changes in the physical and chemicalproperties of hydrogen peroxide vapor under processing conditions andthe decomposition of hydrogen peroxide vapor upon contact with surfacesof various objects within the processing area. As such, undesireddeviations of the concentration of hydrogen peroxide vapor from aprocess set point, along with excessive decomposition of hydrogenperoxide vapor, can result in loss of sterility of the containers andsurrounding aseptic processing area. Moreover, hydrogen peroxide vaporis corrosive in nature, and thus excessive concentration levels ofhydrogen peroxide may bring about detrimental effects to the equipmentin and surrounding the processing area and surfaces of objects withinthe processing area. Furthermore, in accordance with governmentstandards, subsequent use of sterilized containers requires low levelsof residual sterilant.

Heretofore, detection systems for hydrogen peroxide vapor have beenundesirably bulky, as exemplified by conventional near infrared (NIR)analysis apparatus. In addition, current off-line testing methods aretypically too slow for monitoring levels of sterilant with sufficientaccuracy. Previous arrangements have not allowed real time monitoringthroughout an aseptic processing cycle, and in particular, have not beencapable of monitoring concentrations of hydrogen peroxide vapor withinthe sterilization apparatus at select locations along the sterilantsupply system during actual operations. However, U.S. Pat. No. 5,608,156and Taizo et al., “Application of a Newly Developed Hydrogen PeroxideVapor Phase Sensor to HPV Sterilizer”, PDA Journal Of PharmaceuticalScience & Technology, Vol. 52, No. 1/January-February 1998, pp. 13-18,describe methods of detecting the concentration of hydrogen peroxidevapor and an apparatus therefor that appear to address some of theforegoing problems.

The concentration of sterilants detected within a system is generally afunction of various environmental parameters, such as, for example,temperature, relative humidity, and various measurement conditions, suchas, for example, proximate location of measurement. Conventionaldetection systems for sterilant typically cannot or do not account forfluctuations of environmental parameters and measurement conditions.However, such fluctuations can substantially affect the results ofsignal generation and data collection when commercially availablesensors and equipment are used. It is therefore beneficial to maintainoperating parameters proximate the location of measurement as uniform aspossible during data collection.

U.S. Ser. No. 09/443,768, filed Nov. 9, 1999, entitled STERILANTMONITORING ASSEMBLY AND APPARATUS AND METHOD USING SAME, incorporatedherein by reference, describes an integrated system for determining theconcentration of hydrogen peroxide for aseptic process validation,control, and monitoring. This system is compact and can be used foron-line determination of the concentration of hydrogen peroxide. Thesystem requires a unique calibration procedure at regular intervals toguarantee reliable and accurate test results. This system utilizes asensor having elements made of SnO₂. When SnO₂ is heated to a hightemperature, around 400° C., in the absence of oxygen, free electronsflow easily through the grain boundary of the SnO₂ particles. In cleanair, oxygen, which traps free electrons by its electron affinity, isadsorbed onto the surface of the SnO₂ particle, forming a potentialbarrier in the grain boundaries that restricts the flow of electrons,thereby causing the electronic resistance to increase. When the sensoris exposed to hydrogen peroxide vapor, SnO₂ adsorbs its gas moleculesand causes oxidation. This lowers the potential barrier, allowingelectrons to flow more easily, thereby reducing the electricalresistance. Thus, the sensor uses an indirect method to measure theconcentration of hydrogen peroxide vapor.

Voltage data from the output of the sensor must be compared to adatabase derived from a calibration process. The output of two differentsensors cannot be compared directly without calibration. The calibrationprocedure uses several representative points (i.e., concentration at agiven voltage) to establish a mathematical relationship that covers aspecific test window. Only by means of calibration can the outputvoltage of a sensor be converted to a value of concentration.

Calibration procedures are important for minimizing deviations caused bysuch components as semiconductor chips, batteries, and signalconditioning circuits in a sensor in a portable detection system.Calibration procedures are important for minimizing deviations caused bysuch components as temperature and humidity compensation circuits,heating coils, data recording systems, and memory chips in a sensor in afixed detection system.

If the calibration method is not reliable, the concentration of hydrogenperoxide vapor detected by a sensor might be misleading. In turn, anerroneous determination of the concentration of hydrogen peroxide vaporcan bring about contamination in the operation system and result inspoilage. For example, a drop in voltage in the response of the sensorcaused by an increase in the rate of flow of air may be interpreted as adecrease in the concentration of hydrogen peroxide vapor in the system.This apparent decrease may cause the controls in the system to increasethe quantity of hydrogen peroxide delivered, thereby providing anexcessive amount of hydrogen peroxide vapor. An excessive amount ofhydrogen peroxide in the system may result in an excessive amount ofresidue. Conversely, an increase in voltage in the response of thesensor may result from a decrease in the rate of flow of air. If thedelivery rate of hydrogen peroxide is correspondingly reduced, a breachin the sterility of the system may occur.

Calibration of sensors one at a time is inefficient, and, consequently,costly. It is well-known that no two sensors chosen at random are likelyto be identical. Accordingly, it would be desirable to find way tocalibrate individual sensors accurately and at reasonable cost. Inaddition, it would be desirable to find a way to calibrate individualsensors so that one or more of them could be used in portable units. Theuse of a greater number of portable units is desirable so thatmeasurement of the concentration of hydrogen peroxide can be made at anypoint in a production line.

SUMMARY OF THE INVENTION

This invention provides a method and apparatus for calibrating a sensorfor determination of the concentration of a sterilant, e.g., hydrogenperoxide vapor, in a sterilization system.

In one aspect, this invention provides a method for calibrating a sensorthat is used for measuring the quantity of a sterilant in a system fordelivering the sterilant, the method comprising the steps of:

(a) generating reference calibration data, the reference calibrationdata showing a mathematical relationship between a measurable parameter,e.g. voltage, and a quantity of the sterilant, e.g., parts of sterilantper million parts of air (ppm), for a plurality of sensors;

(b) generating sensor calibration data, the sensor calibration datashowing a mathematical relationship between the measurable parameter andthe quantity of the sterilant for an individual sensor; and

(c) normalizing the sensor calibration data to compensate for thedifference between the measurable parameter for the referencecalibration data and the measurable parameter for the sensor calibrationdata, whereby data obtained by the individual sensor can be used toaccurately determine the quantity of sterilant in the system.

The reference calibration data can be generated by a method comprisingthe steps of:

(a) providing a plurality of sensors;

(b) subjecting each of the plurality of sensors to at least twoquantities of air (e.g., 30 cubic meters/hour and 110 cubicmeters/hour), each of the at least two quantities of air having (1) aknown quality (e.g., 60% relative humidity at 70° C.) and (2) a knownconcentration of sterilant vapor (e.g., 10,000 ppm of hydrogen peroxidevapor), the sterilant vapor having a known physical condition (e.g., 70°C.);

(c) measuring the signals (e.g.; voltage) emitted by each of theplurality of sensors, each of the signals being proportional to aconcentration (e.g., ppm) of sterilant vapor;

(d) establishing a mathematical relationship between the signals emittedby each of the plurality of sensors and the concentrations of sterilantvapor for each of the plurality of sensors; and

(e) establishing the reference calibration data by means of astatistical analysis of the signals emitted by each of the plurality ofsensors and the concentrations of sterilant vapor for each of theplurality of sensors.

The sensor calibration data can be generated by a method comprising thesteps of:

(a) providing a sensor;

(b) subjecting the sensor to at least two quantities of air, each of theat least two quantities of air having (1) a known quality and (2) aknown concentration of sterilant vapor, the sterilant vapor having aknown physical condition;

(c) measuring the signals (e.g., voltage) emitted by the sensor, each ofthe signals corresponding to a concentration of sterilant vapor (e.g.,ppm of hydrogen peroxide vapor); and

(d) establishing a mathematical relationship between the signals emittedand the concentrations of sterilant vapor for the sensor.

The sensor calibration data can be normalized to compensate for thedifference between the measurable parameter for the referencecalibration data (e.g., voltage) and the measurable parameter for saidsensor calibration data for the sensor (e.g., voltage) by a methodcomprising the steps of:

(a) selecting a concentration of sterilant vapor;

(b) determining the value of the measurable parameter at which theconcentration of the sterilant vapor obtained from the sensorcalibration data equals the concentration of said sterilant vaporobtained from the reference calibration data; and

(c) adjusting the values measured by the individual sensor a sufficientamount to compensate for the deviation between the reference calibrationdata and the sensor calibration data.

The method of this invention brings about a reduction in the timerequired to calibrate a sterilization system and provides a means fordirectly comparing signals obtained from different sensors so that thecomplex steps involved in the indirect measurement of parameters areavoided.

In one embodiment, which is relatively simple to implement, a linearrelationship between the measured signal (e.g., voltage) and theconcentration of sterilant (e.g., ppm of hydrogen peroxide vapor) isassumed. Points on the curve for an individual sensor (i.e., sensorcalibration data) are moved either vertically or rotationally or both inorder to convert the values of concentration associated with that pointto a value of concentration on the curve representing the referencecalibration data.

The form of the data for calibration of sensors for determining theconcentration of hydrogen peroxide vapor in a stream of air is notcritical. Preferred forms for presentation of data include, but are notlimited to, curves plotted on Cartesian coordinates, nomograms, andlook-up tables containing signal/concentration data.

In one embodiment, in which curves plotted on Cartesian coordinates areemployed, the method involves the steps of

(a) preparing a reference calibration curve, the reference calibrationcurve having a slope and an intercept;

(b) preparing a sensor calibration curve, the sensor calibration curvehaving a slope and an intercept; and

(c) normalizing the sensor calibration curve to compensate for (1) thedifference between the slope of the reference calibration curve and theslope of the sensor calibration curve and (2) the difference between theintercept of the reference calibration curve and the intercept of thesensor calibration curve.

For this embodiment, the reference calibration curve is prepared by amethod comprising the steps of:

(a) providing a plurality of sensors;

(b) subjecting each sensor of the plurality of sensors to at least twoquantities of air, each of the at least two quantities of air having (i)a known quality and (ii) a known concentration of sterilant vapor, thesterilant vapor having a known physical condition;

(c) measuring the signals emitted by each of the plurality of sensors,each of the signals being proportional to the concentration of sterilantvapor;

(d) establishing a linear mathematical relationship between the signalsemitted by each of the plurality of sensors and the concentrations ofsterilant vapor for each of the plurality of sensors; and

(e) establishing the reference calibration curve by means of astatistical analysis of the signals emitted by each of the plurality ofsensors and the concentrations of sterilant vapor for each of theplurality of sensors.

For this embodiment, the sensor calibration curve is prepared by amethod comprising the steps of:

(a) providing an individual sensor;

(b) subjecting the sensor to at least two quantities of air, each of theat least two quantities of air having (1) a known quality and (2) aknown concentration of sterilant vapor, the sterilant vapor having aknown physical condition;

(c) measuring the signals emitted by the sensor, each of the signalsbeing proportional to the concentration of sterilant vapor; and

(d) establishing a linear mathematical relationship between the signalsemitted by the individual sensor and the concentrations of sterilantvapor for the individual sensor.

The sensor calibration curve is normalized to compensate for (1) thedifference between the slope of the reference calibration curve and theslope of the sensor calibration curve and (2) the difference between theintercept of the reference calibration curve and the intercept of thesensor calibration curve by a method comprising the steps of:

(a) determining the intercept of the reference calibration curve;

(b) determining the intercept of the sensor calibration curve;

(c) determining the slope of the reference calibration curve,

(d) determining the slope of the sensor calibration curve;

(e) adjusting the sensor calibration curve, if necessary, in order tocompensate for the difference between the intercept of the referencecalibration curve and the intercept of the sensor calibration curve; and

(g) adjusting the sensor calibration curve, if necessary, in order tocompensate for the difference between the slope of the referencecalibration curve and the slope of the sensor calibration curve.

In another aspect, this invention provides a method for calibrating aportable unit for measuring the concentration of hydrogen peroxide. Inthis method, hydrogen peroxide vapor can be passed through a calibrationvessel, which is submerged in a water bath, under controlled testingconditions. A portable sensor for the detection of hydrogen peroxidevapor installed within the calibration vessel will respond to theconcentration of hydrogen peroxide, the temperature, and the relativehumidity for each test run. The concentration of hydrogen peroxide canbe rechecked by a standard titration method. The accuracy of the valueof the concentration of hydrogen peroxide thus determined can be checkedby means of a detection unit for hydrogen peroxide vapor residue (Dragerkit) to determine the concentration of residual hydrogen peroxide in theflow stream exiting the system. Additional impingers can be added to thesystem to capture all hydrogen peroxide from the stream of flowing airif residual hydrogen peroxide should be detected by a detection unit forhydrogen peroxide vapor residue. Additional data can be generated tocover those conditions that would be expected to be encountered in apractical application.

The method of this invention is capable of conducting calibrations thatmimic both static and dynamic processing conditions, such as seen insterilization of Bosch aseptic machines and hydrogen peroxide spraysterilization of bottles. Flexibility in the selection of parametersencountered in the generation of hydrogen peroxide vapor allows dynamicand static calibration of instruments for the determination of theconcentration of hydrogen peroxide under conditions of high temperature,high concentration, variable rate of flow of air, and variable humidityfor the test cell.

The method of this invention allows the calibration of instruments fordetermining the concentration of hydrogen peroxide vapor for numerousapplications. The combined application of a water bath, a titrationstation, a Drager kit, a metering pump, and a hydrogen peroxide flowmeter allows calibration of a portable detection unit for thedetermination of low concentrations of hydrogen peroxide.

The method of this invention can also be used to investigate thecondensation of hydrogen peroxide by changing the rate of injection ofhydrogen peroxide, the temperature(s) of the heat exchanger(s), thetemperature(s) of the water bath(s), and the rates of flow of air. Therates of condensation and decomposition of hydrogen peroxide can becalculated by determining the difference between the amount of hydrogenperoxide injected and the amount of hydrogen peroxide detected over aselected period of time. The temperature at which air can be saturatedby hydrogen peroxide under known conditions can be estimated as well.

The method of this invention can be used to calibrate a sensor for asterilization system to improve the ability to determine theconcentration of sterilant at a given point in the system at any time atvarious processing stages. By this method, accurate information on theprocess at each stage can be provided.

The method of this invention can also be used to calibrate instrumentsfor monitoring the concentration of hydrogen peroxide vapor in anyenvironment where hydrogen peroxide vapor is employed. Such environmentsinclude, but are not limited to, clean room operations, pharmaceuticalisolator sterilization, aseptic processing systems, and microbiologicalinvestigation of death rate for various bacteria under the effect ofhydrogen peroxide.

This invention provides a method for calibrating instruments fordetermining the concentration of a sterilant, e.g., hydrogen peroxidevapor, for a wide range of processing and operating conditions. Theseoperating condition include, but are not limited to, air temperature,flow rate of air, evaporation temperature, hydrogen peroxide injectionrate, degree of saturation of air by hydrogen peroxide vapor atdifferent temperatures, humidity variations, pressure fluctuations, etc.

It is to be understood that the method of this invention is not limitedto the sterilant hydrogen peroxide. The method of this invention can beused to calibrate instruments for determining the concentration of othergases used for sterilization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram that illustrates a system suitable forgenerating hydrogen peroxide vapor under controllable operatingconditions. This diagram includes the components of the system that areneeded to calibrate a sensor for a sterilization system.

FIG. 2 is a schematic diagram that illustrates a test cell for use incalibration of a sensor for a sterilization system.

FIG. 3 illustrates a type of graph suitable for calibrating a sensor foruse for detecting the concentration of hydrogen peroxide vapor in asystem. The graph includes a reference calibration curve and a sensorcalibration curve.

DETAILED DESCRIPTION

As used herein, the term “sterilant” means a substance that can kill oneor more target microorganisms. The expression “reference calibrationdata” means set of data, derived from examining a plurality of sensors,used to construct a database. The set of data is obtained from at leastthe minimum number of a plurality of sensors that will provide astatistically significant result. The expression “measurable parameter”means a variable that can be measured by a method of measurementacceptable to one of ordinary skill in the art. The term “quantity”means the property or aspect of a thing that can be measured, counted,or compared, such as, for example, rate of flow of air. The expression“sensor calibration data” means set of data, derived from examining anindividual sensor, used to construct a database. The set of data isobtained from a single sensor at a given physical condition and at agiven concentration level. The term “normalizing” means causing thesensor calibration data to conform to the reference calibration data.The expression “air quality” means the physical condition of the airinto which hydrogen peroxide is vaporized. Parameters that can be usedto characterize air quality include, but are not limited to,temperature, relative humidity, pressure, and the like. The term“signal” means a fluctuating electric quantity, as voltage, current,electric field strength, whose variations represent coded information.The expression “mathematical relationship” means a mathematical equationthat expresses at least one parameter as a function of at least oneother parameter. The term “slope” means the rate at which an ordinate ofa point of a line on a coordinate plane changes with respect to a changein the abscissa. The term “intercept” means the distance from the originof coordinates along a coordinate axis to the point at which a line, acurve, or a surface intersects the axis. The expression “Drager kit”means a unit for detecting the concentration of residual hydrogenperoxide vapor. The expression “static condition” refers to thesituation in which both the sensor and the hydrogen peroxide vapor aremomentarily sealed in an air-tight chamber maintained at a constanttemperature and humidity during a test run or calibration run. Theexpression “dynamic condition” refers to the situation in which thesensor is installed in the stream in which the hydrogen peroxide vaporflows.

In one aspect, this invention involves a method for calibrating a sensorthat is used for measuring the quantity of a sterilant in a system fordelivering the sterilant, the method comprising the steps of:

(a) generating reference calibration data, the reference calibrationdata showing a relationship between a measurable parameter, e.g.voltage, and a quantity of the sterilant, e.g., parts of sterilant permillion parts of air (ppm), for a plurality of sensors;

(b) generating sensor calibration data, the sensor calibration datashowing a relationship between the measurable parameter and the aquantity of the sterilant for an individual sensor;

(c) normalizing the sensor calibration data to compensate for thedifference between the measurable parameter for the referencecalibration data and the measurable parameter for the sensor calibrationdata for the individual sensor, whereby the quantity of sterilant in thesystem can be accurately determined.

In order to generate the reference calibration data, each of a pluralityof sensors is subjected to at least two quantities of air (e.g., 30cubic meters/hour and 110 cubic meters/hour). Each of the at least twoquantities of air has (1) a known quality (e.g., 60% relative humidityat 70° C.) and (2) a known concentration of sterilant vapor (e.g.,10,000 ppm of hydrogen peroxide vapor). The sterilant vapor also has aknown physical condition (e.g., 70° C.). The signals emitted by each ofthe sensors are measured. Each of the signals measured corresponds tothe concentration of sterilant vapor. Then, a mathematical relationshipis established between the signals measured and the concentrationscorresponding to the measured signals for each of the sensors. Theforegoing operations are repeated until a mathematical relationshipbetween the signals measured and the concentrations corresponding to themeasured signals has been established for each sensor of the pluralityof sensors. Then, the reference calibration data is derived from thedata and mathematical relationships obtained from the foregoingmeasurements. Typically, the reference calibration data represents themean (average) of the data obtained from the foregoing measurements. Thenumber of sensors used to prepare the reference calibration data ispreferably sufficient to provide reference calibration data thatexhibits a normal distribution with about 95% of the values of thesensors falling within two standard deviations of the mean. In general,it is preferred that at least 10 sensors be used to provide data forpreparing the reference calibration data.

In order to generate the sensor calibration data, an individual sensoris provided. This sensor is subjected to at least two quantities of air,each of the at least two quantities of air having (1) a known qualityand (2) a known concentration of sterilant vapor. The sterilant vaporhas a known physical condition. The signals emitted by the sensor aremeasured. Each of the measured signals corresponds to a concentration ofsterilant vapor. A mathematical relationship is established between thesignals emitted by the sensor and the concentrations corresponding tothe signals measured for the sensor. Then, the sensor calibration datais derived from the data and mathematical relationships obtained fromthe foregoing measurements

Then the sensor calibration data is normalized to compensate for thedifference between the measurable parameter for the referencecalibration data (e.g., voltage) and the measurable parameter for thesensor calibration data for the individual sensor. In order to carry outthe normalization step, a concentration of sterilant vapor is selected.The value of the measurable parameter at which the concentration of thesterilant vapor obtained from the sensor calibration data equals theconcentration of said sterilant vapor from the reference calibrationdata is determined. This value of the measurable parameter for a givenconcentration must be in both the reference calibration data and thesensor calibration data. The values of the measurable parameter in thesensor calibration data are adjusted a sufficient amount to compensatefor the deviation of the sensor calibration data from the referencecalibration data.

The form of the data for both the reference calibration data and thesensor calibration data is not critical. Preferred forms of datainclude, but are not limited to, curves plotted on Cartesiancoordinates, nomograms, and tables containing signal/concentration data.Of course, the form for the reference calibration data and the sensorcalibration data must be the same in order to ensure a meaningfuladjustment.

In one embodiment, in which curves plotted on Cartesian coordinates areemployed, the calibration can be carried out in the following manner. Alinear reference calibration curve having a slope and an intercept isprepared. The value of the concentration is plotted on the abscissa andthe value of signal is plotted on the ordinate. A linear sensorcalibration curve for an individual sensor having a slope and anintercept is prepared. The value of the concentration is plotted on theabscissa and the value of signal is plotted on the ordinate. Thereference calibration curve and the sensor calibration curve intersectat a set of coordinates. The sensor calibration curve is normalized tothe reference calibration curve to compensate for (1) the differencebetween the slope of the reference calibration curve and the slope ofthe sensor calibration curve and (2) the difference between theintercept of the reference calibration curve and the intercept of thesensor calibration curve. Normalization can be carried out in severalways. In one embodiment of normalization, the sensor calibration curvecan be adjusted physically, by adjusting it vertically and rotationallyso that the intercept and the slope of the sensor calibration curveconform to the intercept and the slope, respectively, of the referencecalibration curve. In another embodiment of normalization, the variablerepresenting the signal on the sensor calibration curve (i.e., voltageor log (voltage)) can be substituted into the equation that relates theconcentration and the signal on the reference calibration curve.

Referring now to FIGS. 1 and 2, compressed air at room temperature isintroduced into a system 10 suitable for generating hydrogen peroxidevapor under controllable operating conditions by means of a valve 12.The compressed air is represented by the letter “A”. The air is filteredby means of an air filter 14 to remove dirt and other foreignsubstances. A valve 16, preferably a diaphragm valve, is used to controlthe flow rate of the air, and a pressure regulator 18 is used to adjustthe pressure of the air. The rate of flow of the air is monitored by aflow meter 20 and the parameters of air quality are measured andrecorded by a data acquisition system (e.g., Keithley 500 dataacquisition system, Keithley Instruments, Inc., 28775 Aurora Rd.,Cleveland, Ohio 44139). The parameters of air quality includetemperature, humidity, and pressure. These parameters are monitored by atemperature sensor 22, a humidity sensor 24, and a pressure sensor 26,respectively. Because of seasonal variations in the quality of air inthe room containing the components of the system 10, the moisturecontent of the air is adjusted to a predetermined level for the purposeof the calibration. The moisture content of the air is adjusted by usingeither a dehumidifier 28, which is operated by opening or closing avalve 30, or a humidifier 32, which is operated by opening or closing avalve 34. The adjusted air quality is measured by means of a temperaturesensor 36 and a humidity sensor 38 located downstream of the exit port40 of the dehumidifier 28 and the exit port 41 of the humidifier 32.Accordingly, the temperature level and the humidity level of the airwill be known.

The air is then heated to a specified temperature by means of a heatexchanger 42. The concentration of hydrogen peroxide (hereinafter. H₂O₂)vapor in a sample of air saturated with H₂O₂ vapor is a function oftemperature. As the temperature of the air is increased, the air canhold a higher concentration of H₂O₂ vapor. The temperature of the aircan be monitored by a temperature sensor 44 located downstream of theexit 46 of the heat exchanger 42. In the heat exchanger 42, temperatureis a function of steam pressure. By adjusting the steam pressure of theheat exchanger 42, the temperature of the air can be controlled. The airis then introduced into a heat exchanger 48 and merged with a stream ofH₂O₂ droplets created by an atomizer 50. The H₂O₂ is caused to evaporaterapidly and is further heated to a higher temperature. A metering pump52 controls the rate of injection of liquid H₂O₂ into the air flowinginto the heat exchanger 48. A flow meter 54 is used to monitor the flowrate of liquid H₂O₂ to ensure that the rate of injection of H₂O₂ iscorrect. The temperature of the liquid H₂O₂ can be measured by atemperature sensor 56 to ensure a consistent temperature for allcalibration operations. A tank for storing the liquid H₂O₂ is designatedby the reference numeral 58, and a valve for controlling the injectionof liquid H₂O₂ into the stream of air is designated by the referencenumeral 60. The tank 58 and the valve 60 are those conventionally usedfor introducing liquid H₂O₂ into a sterilization system. Accordingly,the tank 58 and the valve 60 are resistant to H₂O₂.

The mixture of air and H₂O₂ vapor may be in one of several conditions.These conditions may be described as follows:

1. Air is saturated with H₂O₂ vapor—condensation of droplets of the H₂O₂vapor occurs when temperature decreases;

2. Air is not saturated with H₂O₂ vapor—H₂O₂ vapor decomposes on thesurfaces of certain materials; H₂O₂ vapor condenses when temperaturedrops below the saturation temperature;

3. Under both conditions (saturated and unsaturated), if the temperatureincreases, H₂O₂ droplets will revaporize;

4. Latent heat of evaporation of H₂O₂ is positive; evaporation of H₂O₂generates extra heat in the system.

The temperature of the air is generally higher than the temperature atwhich the heat exchanger 48 is set, and the temperature of the air isdifficult to predict and control.

Two additional heat exchangers 62 and 64 can be used to reduce thetemperature of the air and ensure that the air is saturated with H₂O₂vapor at a known temperature. For the air to reach a state ofsaturation, it is common to evaporate a liquid at a high temperature andthen reduce the temperature. If a sufficient quantity of liquid isinjected into the system at a high temperature, saturation can beguaranteed when the vapor is cooled. Also, the saturation process shouldbe conducted at the last possible point before the H₂O₂ vapor is to beused for sterilization, because it is difficult to control condensationand decomposition at some other point in the system.

The temperature of the air at various locations in the system 10 can bemonitored by temperature sensors 65 a, 65 b, and 65 c.

The air containing the H₂O₂ is then introduced into a test cell 66. Asimplified diagram of the test cell 66 is shown in FIG. 2. The test cell66 is described in U.S. Ser. No. 09/443,768, filed Nov. 19, 1999,entitled STERILANT MONITORING ASSEMBLY AND APPARATUS AND METHOD USINGSAME, incorporated herein by reference. In U.S. Ser. No. 09/443,768, thetest cell is referred to as a sterilant monitoring assembly and isdesignated by the reference numeral 200. A sensor 68 suitable fordetermining the concentration of H₂O₂ vapor is installed in the testcell 66 in order to be calibrated. It is preferred that the conditionswithin the test cell 66 be substantially identical to those used in anactual sterilization process. It should be noted that the data for thecalibration operation is likely to be valid only after the system 10reaches a state of equilibrium. Accordingly, the system 10 should be runa sufficient amount of time in order to reach a state of equilibrium.

It is preferred that two types of runs be performed in each calibrationoperation. In a static test run, an internal cell 70 in the test cell 66is placed in fluidic communication with the flowing stream of air for aspecified amount of time, the flowing stream of air having reached astate of equilibrium. In order to determine the concentration of H₂O₂vapor in a sample of air, the internal cell 70 in the test cell 66 isthen sealed by means of a piston 72 moving in a cylinder 74 under theforce of compressed air pushing against a spring 76. The compressed aircan be introduced to the cylinder 74 through an inlet 78. AnH₂O₂-resistant O-ring 80 can be used to ensure that the internal cell 70is sealed against a heat isolation plate 82. The cover of the testchamber 66 is designated by the reference numeral 84 and a thermocoupleis represented by the reference numeral 86. The data obtained by meansof this static test run reflects the level of concentration of H₂O₂vapor, independent of the rate of flow of air. The flowing airsurrounding the internal cell 70 serves as an insulating jacket tomaintain a uniform and constant temperature during the static test run.In a dynamic test run, the internal cell 70 is placed in fluidiccommunication with the flowing air at all times. In other words, theinternal cell 70 is not sealed against the heat isolation plate 82. Inthe dynamic test run, the data obtained only reflect the concentrationof H₂O₂ vapor at a specific flow rate of air.

The results obtained from the two types of calibration runs can be usedto verify the determination of the concentration of H₂O₂ vapor underactual sterilization conditions. For example, if the readings ofconcentration of H₂O₂ vapor determined in static test runs remainsubstantially constant, but if the readings of concentration of H₂O₂vapor determined in dynamic test runs vary substantially, (1) the flowrate of the air may have changed, (2) the rate of addition of sterilantto the system may have changed, or (2) something may be wrong with thesystem. Similarly, if the readings of concentration of H₂O₂ vapordetermined in static runs conditions vary substantially, but if thereadings of concentration of H₂O₂ vapor determined in dynamic test runsremain substantially constant, (1) the flow rate of the air may havechanged, (2) the rate of addition of sterilant to the system may havechanged, or (2) something may be wrong with the system. Assuming thatthe flow rate of the air has not changed, and that the rate of additionof sterilant to the system has not changed, what may be wrong with thesystem is that the sensor for determining the concentration of H₂O₂vapor is providing unreliable readings, which may be the result ofunreliable calibration. If the method for calibrating the sensor isunreliable, the concentration of H₂O₂ vapor determined by the sensor maybe misleading. An erroneous determination of the concentration of H₂O₂vapor can result in contamination of the system and result in spoilageof product. For example, a drop in voltage in the response measured bythe sensor caused by an increase in the rate of flow of air may beinterpreted as a decrease in concentration of H₂O₂ vapor in the system.This apparent decrease in response may cause controls in the system toincrease the quantity of H₂O₂ delivered, thereby providing an excessiveamount of H₂O₂ vapor. An excessive amount of H₂O₂ in the system willresult in an excessive amount of residue. Conversely, an increase involtage in the response of the sensor may result from a decrease in therate of flow of air. If the delivery rate of H₂O₂ is correspondinglyreduced, a breach in the sterility of the system may occur. For theforegoing reasons, it is frequently desirable to cross-check calibrationruns of sensors.

As stated previously, the system 10 should be run a sufficient amount oftime in order to reach a state of equilibrium. When the system isactivated initially, the air exiting the test cell 66 flows into asample collection port 88 positioned downstream from the test cell 66. Avalve 90 is operated to allow the air flowing through the samplecollection port 88 to be transported to a catalytic converter 92, whichaccelerates the decomposition of H₂O₂ (i.e., breakdown of H₂O₂) beforeit enters the ambient environment. During this initial activationperiod, a valve 94, preferably a programmable three-way valve, allowsair to flow through the system 10 to a valve 96, preferably a three-wayvalve, and from there to the catalytic converter 92. During this initialactivation phase, a valve 98, which will be described later, is closed.

After the initial activation phase, i.e., when the system 10 has reacheda state of equilibrium, the signal (e.g., voltage data) obtained fromthe sensor 68 is correlated with the concentration of H₂O₂ vapor in thetest cell 66. In order to perform this correlation, a sample of aircollected via the sample collection port 88 is passed through atitration station 100, which comprises a series of impingers 102 set inan ice bath 104. The flow rate of the sample of air is measured by aflow meter 106; the duration of the test is precisely controlled by theoperation of the valve 94. In order to perform this measurement, thevalve 94 allows air to flow through the system 10 to the valve 96, whichis operated to allow the air to enter the titration station 100. Thevalve 90 is operated to allow air to flow to the catalytic converter 92,and the valve 98 is closed. The amount of H₂O₂ captured in the impingers102 is a function of time. By measuring the time consumed and the amountof H₂O₂ collected in the impingers 102, the concentration of H₂O₂ can beestimated. For example, if the flow of air is 10 cubic meters perminute, and if approximately 10 mL of H₂O₂ vapor condense in theimpingers 102, then the concentration of H₂O₂ is equal to approximately1 mL per cubic meter. The rate of flow of the sample of air to thetitration station 100 can be controlled by adjusting the size of anorifice in the sample collection port 88.

When the titration station 100 is used to determine the concentration ofH₂O₂ in the sample of air in the test cell 66, the valve 94 is operatedto allow the air containing the H₂O₂ vapor to bypass a test chamber 108in a water bath 110. The test chamber 108 can be used to calibrate aportable H₂O₂ detection unit 112. A portable H₂O₂ detection unit is onethat can be installed in a bottle sterilizer, such as, for example, aBosch apparatus, or that can be placed at any location in an isolator ora clean room to monitor sterilization by H₂O₂. A portable H₂O₂ detectionunit can also be installed inside a container, such as a 32-ounceplastic container, to detect the H₂O₂ vapor condition after an H₂O₂vapor spraying procedure for sterilization of bottles. The portable unitis an integrated unit comprising an H₂O₂ sensor, a data recordingelement, a battery for power supply, a signal conditioning circuit, anear infrared (NIR) receiver, a DC/DC converter, and a temperaturesensor. Portable units are preferably programmable, and the dataobtained by using such a unit can downloaded after a test. A portableunit has a size of about 1 inch by 2 inches by 2 inches. Portable H₂O₂detection units are described in U.S. Ser. No. 09/360,772, filed Jul.26, 1999, entitled SELF-CONTAINED STERILANT MONITORING ASSEMBLY ANDMETHOD OF USING SAME, incorporated herein by reference. Beforemeasurements are performed in the test chamber 108, it is preferred thatthe system 10 be run for a sufficient amount of time to avoidcondensation of H₂O₂. Such condensation results from the temperaturedifferential between the air containing the H₂O₂ vapor and the internalsurfaces of pipes, the test cell 66, the test chamber 108, and thevalves. During the initial activation phase that is suggested prior toutilizing the test chamber 108, the valve 94 is operated to allow air toflow into the test chamber 108, and the valve 98 is open. The valve 96is operated to allow the air that exits from the test chamber 108 toflow to the catalytic converter 92, and the valve 90 is open. When thetest chamber is used to calibrate a portable detection unit 112, thevalve 96 is operated to prevent the air that exits from the test chamber108 from flowing to the catalytic converter 92, and the valve 90 isopen. The valve 96 is operated to allow the air that exits from the testchamber 108 to flow to the titration station 100.

During the calibration of the portable unit 112, the portable unit 112is placed inside the test chamber 100 and operated by a remote control(not shown), preferably a near infrared (NIR) remote control. The flowof air is adjusted by adjusting the size of the orifice of the samplecollection unit 88. The temperature of the water bath 110 is the same asthe temperature of the sample of air containing the H₂O₂ vapor. Bymeasuring the duration of the run and the flow rate of the sample ofair, the concentration of H₂O₂ vapor in the sample can be calculated inthe same manner as was used to calibrate the sensor 68 tested in thetest cell 66. A relationship between the voltage data obtained from theportable H₂O₂ detection unit 112 and the concentration value of H₂O₂vapor can then be established.

The valve 90 is allowed to remain open most of the time, but isgenerally closed when a low flow rate of air is required. If the flowrate of air is low, the valve 90 is closed and the air will flow onlythrough the valves 94, 96, and 98. It should be noted that the valves12, 16, 30, 34, and 60 are operated as required in order to set theconditions of flow rate and humidity and vary the quantity of H₂O₂admitted to the system 10.

Additional temperatures 114 a, 114 b, and 114 c can be used to monitortemperature in the test chamber 108 and in the titration station 100. Apressure sensor 116 can be used to monitor pressure in the test chamber108. A Drager kit 118 can be used to detect the concentration ofresidual H₂O₂ vapor that exits the system 10.

The components of the system including, but limited to air conditioningcomponents (e.g., pressure regulators, flow meters, dehumidifier(s),humidifier(s), heat exchangers), temperature sensors, humidity sensors,atomizer, metering pump, test cell, water bath, test chamber, titrationstation, and valves, are all commercially available. Optimal selectionof these components can be made by one of ordinary skill in the art.While the sizes of the system 10 and the components thereof are notcritical, the dimensions of a few components are given to provide anidea of the amount of space required for a calibration system. The sizeof the system 10 is about 3 meters×4 meters×3 meters. The size of thetest cell 66 is about 1 foot×0.5 foot×0.5 foot. The size of the portableunit 112 is about 1 inch×2 inches×2 inches. The size of the Drager kit118 is about 3 inches×3 inches×4 inches. The size of the test chamber108 is about 8 inches high×about 4 inches in diameter. The volume of animpinger 102 is about 125 mL.

The operating conditions of the system can vary but they generally fallwithin the following ranges:

(a) temperature of the air ranges from about −10° C. to about 85° C.;

(b) relative humidity of the air ranges from about 10 to about 100%;

(c) the flow rate of air ranges from about 1 to about 200 cubic metersper hour;

(d) the concentration of sterilant ranges from 100 to 25000 ppm.

Calibration procedures should be conducted at regular time intervals toobtain reliable and accurate test results. The calibration system 10 canensure the reliability and accuracy of the prediction of concentrationof H₂O₂ determined by an H₂O₂ sensor by simulation of processconditions.

A series of calibration runs can be conducted for different temperatureranges, various H₂O₂ concentration levels, selected flow rates, adjustedrelative humidity, different liquid H₂O₂ concentrations.

The method of this invention can be used to calibrate a sensor for asterilization system to improve the ability to determine theconcentration of sterilant at a given point in the system at any time atvarious processing stages. By this method, accurate information on theprocess at each stage can be provided.

The method of this invention can also be used to calibrate instrumentsfor monitoring the concentration of H₂O₂ vapor in any environment whereH₂O₂ vapor is employed. Such environments include, but are not limitedto, clean room operations, pharmaceutical isolator sterilization,aseptic processing systems, and microbiological investigation of deathrate for various bacteria under the effect of hydrogen peroxide.

This invention provides a method for calibrating instruments fordetermining the concentration of a sterilant, e.g., H₂O₂ vapor, for awide range of processing and operating conditions. These operatingcondition include, but are not limited to, air temperature, flow rate ofair, evaporation temperature, hydrogen peroxide injection rate, degreeof saturation of air by hydrogen peroxide vapor at differenttemperatures, humidity variations, pressure fluctuations, etc.

It is to be understood that the method of this invention is not limitedto the sterilant hydrogen peroxide. The method of this invention can beused to calibrate instruments for determining the concentration of othergases used for sterilization.

The following non-limiting example further illustrates the method ofthis invention.

EXAMPLE 1

The following example illustrates the calibration of a sensor by themethod of this invention.

The system 10 shown in FIG. 1 was used to carry out the method of thisexample. When the system is activated, the valves 12, 16, 30, 34, and 60are operated so as to deliver the desired quantities of air and H₂O₂ andthe desired quality of air to the test cell 66. Because the test cell 66is being used to calibrate a sensor 66, the valves 94, 96, and 98 areoperated so that the air bypasses the test chamber 108. The valve 90 isallowed to remain open during the calibration runs of this example.Typical air quality conditions are 65% relative humidity, 70° C. Typicalflow rates of air range from about 30 cubic meters/hour to about 110cubic meters/hour.

Reference Calibration Data

Fifteen (15) sensors (TGS 816, Figaro USA, Inc., Glenview, Ill. 60025)were used to prepare a linear reference calibration curve. This curve isdescribed by the equation y₁=a₁+b₁x, where x represents concentrationsof H₂O₂, y₁ represents the mean of the voltage readings taken at aparticular value of concentration of H₂O₂ for the 15 sensors, a₁represents the mean (average) of the intercepts of the plots of voltagev. concentration of H₂O₂ for the 15 sensors, and b₁ represents the mean(average) of the slopes of the plots of voltage v. concentration of H₂O₂for the 15 sensors. See FIG. 3. The value of the slope and the value ofthe intercept of each of the 15 sensors were determined by datacollected at at least three points (concentrations) on the abscissa.Each point was determined by at least two repetitions at eachconcentration. Table 1 is a compilation of the slopes and intercepts foreach of the 15 sensors used to prepare the reference calibration data.

TABLE 1 Sensor Intercept Slope 1 −0.96 0.39 2 −0.954 0.36 3 −0.938 0.364 −1.11 0.41 5 −0.95 0.361 6 −1.04 0.37 7 −1.2 0.34 8 −1.04 0.37 9 −1.30.342 10 −1.2 0.45 11 −1.4 0.34 12 −1.11 0.44 13 −0.98 0.361 14 −1.120.41 15 −1.05 0.38

The data in Table 1 yielded a mean slope value (i.e., mean of b₁) of0.379 and a mean intercept value (i.e., mean of a₁) of −1.090. Thefollowing table, Table 2, shows the relationship between voltage andconcentration of H₂O₂ for reference calibration data utilizing the slopevalue of 0.379 and the value of −1.090, which were derived from the datain Table 1.

TABLE 2 Concentration of Voltage Log (concentration Log H₂O₂ (ppm)(volts) of H₂O₂) (voltage) 3535 1.838 3.548 0.254 5839 2.210 3.766 0.3378250 2.510 3.916 0.394 10607 2.715 4.026 0.435 12964 2.898 4.113 0.468

Sensor Calibration Data

One (1) sensor (TGS 816, Figaro USA, Inc., Glenview, Ill. 60025) wasused to prepare a linear sensor calibration curve. This curve isdescribed by the equation y₂=a₂+b₂x, where x represents concentrationsof H₂O₂, y₂ represents the voltage reading taken at a particular valueof concentration of H₂O₂ for a single sensor, a₂ represents theintercept of the plot of voltage v. concentration of H₂O₂ for the singlesensor, and b₂ represents the slope of the plot of voltage v.concentration of H₂O₂ for the single sensor. See FIG. 3. The values ofthe slope and the intercept of the single sensor were determined by datacollected at at least three points on the abscissa. Each point wasdetermined by at least two repetitions at each concentration. Theconcentrations used are shown in Table 3.

TABLE 3 Concentration of Voltage Log (concentration Log H₂O₂ (ppm)(volts) of H₂O₂) (voltage) 3535 1.838 3.548 0.264 5839 2.210 3.766 0.3448250 2.510 3.916 0.400 10607 2.715 4.026 0.434 12964 2.898 4.113 0.462

The data in Table 3 yielded a slope value of 0.351 and an interceptvalue of −0.098.

Normalizing

The following mathematical relationships were used to convert readingsfrom an individual sensor to standardized values based on the referencecalibration data. In the equations that follows, “voltage_(r)” meansvoltage of a point on the reference calibration curve, “voltage_(s)”means voltage of a point on the sensor calibration curve,“concentration_(r)” means concentration of a point on the referencecalibration curve, and concentration_(s)” means concentration of a pointon the sensor calibration curve.

Reference calibration curve:

Log (voltage_(r))=−1.090+0.379 Log (concentration_(r))

Sensor calibration curve:

Log (voltage_(s))=−0.981+0.352 Log (concentration_(s))

Log (concentration_(s))=[Log (voltage_(s))+0.981]/0.352

Substituting the equivalent of Log (voltage_(s)) into the equation forLog (voltage_(r)) yields:

Log (voltage_(r))=−1.090+0.379×[Log (voltage_(s)]+0.981]/0.352

Thus, one can convert voltage data obtained from an individual sensor toa value of concentration on the reference curve. In order to compareseveral sensors, Table 4 shows values encountered in a typicalconversion from the value of output voltage of a sensor to a value ofvoltage on the reference calibration curve, which value corresponds to aspecific concentration of H₂O₂ vapor.

TABLE 4 Log Log Concen- (con- (voltage tration centra- Log Log convertedof tion of Voltage (voltage- Voltage (voltage- from volt- H₂O₂ H₂O₂)(ref¹) ref) (sen²) sen) age (sen)) 6000 3.778 2.196 0.342 2.224 0.3470.342 8000 3.903 2.448 0.389 2.461 0.391 0.389 10000 4.000 2.664 0.4262.662 0.425 0.426 12000 4.079 2.855 0.456 2.838 0.453 0.456 14000 4.1463.027 0.481 2.996 0.477 0.481 16000 4.204 3.184 0.503 3.140 0.497 0.503¹The values of voltage on the reference calibration curve correspond toconcentrations obtained by using an equation derived from statisticalanalysis. The equation was a linear equation in which the slope and anintercept of a line could be determined. ²The values of voltage wereobtained by an experiment in which the value of voltage corresponded tothe concentration of H₂O₂.

The seventh column of Table 4 shows that for a given concentration ofH₂O₂, the value of log (voltage) obtained by means of the method of thisinvention is equal to the value of log (voltage) of the referencecalibration curve, as shown in the fourth column of Table 4. Thus, if anindividual sensor give a voltage reading of 0.347 volt, the reading willbe adjusted to 0.342 volt, which translates to a concentration of H₂O₂of 6000 ppm. The method of this invention allows the use of a pluralityof sensors to measure the concentration levels of sterilant by means offixed reference calibration data, thereby reducing the number of dataconversions encountered when numerous sensors are used. This feature isextremely useful when several sensors are used at the same time underactual sterilization conditions.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

What is claimed is:
 1. A method for calibrating a portable unit formeasuring the concentration of a sterilant, said method comprising thesteps of: (a) passing a sterilant vapor through a vessel, said vesselbeing submerged in a water bath, said portable unit being installedwithin said vessel, said portable unit responsive to the concentrationof sterilant, the temperature, and the relative humidity for a test run;and (b) determining the concentration of said sterilant by a titrationmethod.
 2. The method of claim 1, wherein said sterilant is hydrogenperoxide.